Journal of Agricultural Science and Technology A 5 (2015) 30-39 doi: 10.17265/2161-6256/2015.01.005 D DAVID PUBLISHING

Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic

Nataliya Vladimirovna Punina1, Mikhail Anatolievich Remnev2 and Alexey Fedorovich Topunov1 1. A. N. Bach Institute of Biochemistry RAS, Moscow 119071, Russia 2. The Federal State Unitary Enterprise All-Russia Research Institute of Automatics, Moscow 127055, Russia

Abstract: Rapid and reliable diagnostics and identification of pathogenic and symbiotic bacteria are at the top of the agenda. In the first case, they are important to control and prevent crop damages, and thus reduce economic losses. In the second, it’s necessary to design and monitor quality of biofertilizer to raise its effectiveness and crop capacity. Development of accurately, rapidly, technically and commercially accessible methods remains a critical problem for the bacteria with comprehensive phylogenetic structure. In this work, we investigated pathogenic Xanthomonas and Ralstonia and symbiotic . The aim of this investigation was to examine the applicability of the novel methods for phylogenetic study, identification and diagnostics of closely related species of these genera. The conventional phenotypic and genotypic (16S rRNA, gyrB) methods were applied as referents. Novel polymerase chain reaction (PCR)-based approaches, single-adapter amplified fragment length polymorphism (saAFLP) and comparative analyses of hin-region and Xcc0006-0007 sequences, were first employed for the investigations. Phenotypic tests, 16S rRNA and gyrB analysis distinguished bacteria at the genus level, but failed to identify them to the species robustly. The new methods identified bacteria at the inter-species level more precisely. This identification agreed with the accepted genera’s classifications. The only exceptions were X. fuscans & X. citri and X. perforance & X. euvesicatoria which clustered together. The further outcome of this study was achieved hin-region-based genus-specific PCR primers for the express-diagnostics of the genera. Summary, these new methods can be applied for genome-based phylogeny investigations and as convenient and accurate tools for identification and routine laboratory diagnostics of these comprehensive genera.

Key words: Hin-region, saAFLP, Xanthomonas, Rhizobium, Sinorhizobium, Ralstonia.

1. Introduction beneficial associates and symbiotic microbes for biofertilizer design; (3) to provide surveillance and Plan-associated bacteria share a long history with control for both pathogenic and beneficial bacteria, plants and profound impact on the health and and thus prevent a bacterial diseases and increase a agricultural yield of crop [1]. There are two main crop yield. groups of bacteria based on their adverse/beneficial For a long time, bacterial classification has been effects on the host plants: pathogens and mutualists based largely on culture and biochemical tests, [2]. Investigation of the plant-bacterial interactions DNA-DNA hybridization, and 16S rRNA gene and microorganism’s taxonomic classification are the sequencing analysis [3]. Nowadays, classification, most important and developing directions of the identification and diagnostics integrate also new present biology [1]. The received knowledge can be techniques to determine both phenotypic and used in agricultural and biotechnological sectors: (1) genotypic characteristics [3-7]. The use of multilocus to diagnose most prevalent and most economically sequence typing (MLST), multilocus sequence important pathogens and prevent plant diseases; (2) to analysis (MLSA) and protein-typing are becoming identify and describe most common groups of very important for delineation of plant pathogenic and

Corresponding author: Nataliya Vladimirovna Punina, symbiotic bacteria at species level and lower level [8]. Ph.D., research fields: microbiology and molecular biology. Over the last few years, these methods were improved E-mail: [email protected].

Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria 31 dramatically: increasingly mechanized to raise speed, methods were applied before [28]. There is still a need discrimination power and throughput, and to reduce for rapid, accurate, easy and low-cost approaches that cost of the laboratory analysis [9-16]. However, every can be applied to production for identification (control method has its limitations and selection of the and monitor) of the industrial strains. integrated approaches significantly depends on the In this work, the new DNA marker hin-region and target microorganism, time limits, assay accuracy and single-adapter amplified fragment length geographical scale [3, 17]. These methodical polymorphism (saAFLP) analysis were first used to disadvantages has become more complicated due to identify and describe the closely-related strains of the globalization, climate change, increased human genera Xanthomonas, Ralstonia and Sinorhizobium. mobility and bacterial evolution. Therefore, there is a Obtained results were compared with data derived by strong need to develop more rapid, accurate and the traditional pathogenicity and biochemical, universal techniques and methods to identify and comparative analysis of 16S rRNA, gyrB and detect economically important bacteria. Xcc0006-0007 nucleotide sequences. The potential of In this work, we studied two kinds of bacteria new methods for identification and diagnostics of characterized by the type of nutrition and agricultural strains was evaluated for these genera. importance: phytopathogenic genera Xanthomonas 2. Materials and Methods and Ralstonia and symbiotic Sinorhizobium. Xanthomonas and Ralstonia affect more than 400 2.1 Bacterial Strains species of agricultural crops, causing bacteriosis and The original and type strains from weeds and thus leading to major economic losses [18, 19]. Their agricultural crops were used in this study. One taxonomic structures are intricate and are being hundred and fourteen strains of Xanthomonas spp. constantly revised [20-22]. These genera had been were provided from All-Russian collection of subjected to numerous studies to clarify the microorganisms, G. K. Skryabin Institute of and phylogeny, although all applied methods were Biochemistry and Physiology of Microorganisms partially sufficient for their identification and (VKM IBPM RAS; Pustchino, Russia), and Zabolotny diagnostics [21-27]. The disadvantage of the present Institute of Microbiology and Virology, National diagnostics of these genera is testing of the strain at Academy of Sciences of Ukraine (IMV; Kiev, the one taxonomic level and only a certain known Ukraine). Twelve Ralstonia sp. strains were isolated group of bacteria. Therefore, the development of new from wilted tomato and potato in Russia. The known rapid and efficient methods is still crucial for detection cultures of these genera obtained from the and identification of these phytopathogens. International Collection of Phytopathogenic Bacteria The genus Sinorhizobium (Ensifer) contains bacteria (ICPB; Davis, CA, USA), American Type Culture capable to fixate nitrogen in symbiosis with leguminous Collection (ATCC; Manassas, VA, USA) and plants including agricultural crops, e.g., [28]. National Collection of Plant-Pathogenic Bacteria This genus contains closely-related species, and some (NCPPB; York, England) were also included for of them are prospective for biofertilizer reference. Two referential strains of nitrogen-fixing production [29]. Identification and characterization of Sinorhizobium meliloti B-117 and B-1009 were this genus are necessary to reveal and select most received from the collection of VKM IBPM RAS. efficient symbiotic bacteria, create most productive Nine strains of Sinorhizobium spp. were isolated from plant-symbiont combination and thus increase crop root nodules of field-grown soybeans in Russia in capacity of fields. Different phenotypic and genetic 2013.

32 Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria

2.2 DNA Isolation were amplified and sequenced using previously constructed primers UP1/UP2r and protocols [35]. The overall cellular DNA specimens were isolated from strains cultured on agarized yeast extract and 2.7 PCR Amplification and Sequencing of the tryptone (TY) medium (g/L): 5.0 g/L yeast extract, Xcc0006-0007 Region 10.0 g/L bacto-tryptone, 10.0 g/L NaCl and 20.0 g/L Nucleotide sequences of the Xcc0006-0007 region agar. DNA was isolated from cells on days 1-2 of of Xanthomonas spp. were amplified and sequenced culture by sorption onto magnetic particles (“Silex”, according previously published protocol [36]. Russia). 2.8 saAFLP Analysis 2.3 Phenotypic Characterization We modified amplified fragment length The morphological and biochemical characteristics polymorphism (AFLP) method developed before by of the pure bacterial cultures were determined based Vos et al. [37] and named new modification as on the general strategy of phenotypic differentiation saAFLP [38, 39]. The phylogenetic relationships described in Refs. [30-32]. between closely related strains Bacilllus cereus group 2.4 Pathogenicity Identification: Plant Nodulation Tests and Rhizobium leguminosarum had been successfully analyzed using this method [38, 39]. The procedure of All strains of genera Xanthomonas and Ralstonia saAFLP comprises three steps: (1) treatment of the were tested for their pathogenicity on wide host range using previously described methodology [33]. extracted bacterial DNA by one of the genus specific Four-week-old seedlings (two- to three-leaf-stage) of restriction endonucleases and ligation with the single tomato, cabbage, pepper, geranium and tobacco were stranded adapter; (2) PCR amplification with the inoculated by leaf and steam inoculation methods. The single primer which is complementary to the adapter incubated plants were then kept on a greenhouse sequence; (3) electrophoretic separation of the PCR bench until symptoms development. Each experiment products in agarose gel. The fundamentally new was repeated three times. Results were recorded 7 d aspects of this methodological procedure include: (1) and 16 d after inoculation. performing restriction analysis and ligase reaction in the same tube concurrently; (2) selecting restriction 2.5 Polymerase Chain Reaction (PCR) Amplification endonucleases by using computational analysis and Sequencing of the 16S rRNA Gene (“ReVer”, Remnev and Punina, 2014, unpublished) so The PCR analysis and subsequent determination of that we can obtain 10-30 DNA fragments after the the 16S rRNA nucleotide sequences were conducted treatment; (3) using the one single-stranded adapter. using the universal primers and protocols as described 2.9 PCR Amplification and Sequencing of Hin-Region previously [34]. All amplified fragments were separated by an electrophoresis on 1.5% agarose gel. Hin-region amplification was carried out using Sequencing was carried out using genetic analyzer primers designed specifically for the genera 3130xl, “Applied Biosystems” (ABI) automated Sinorhizobium, Ralstonia and Xanthomonas. The sequencing machine (USA). primers and PCR amplification protocols were patented for the genera Rhizobium and Xanthomonas [40]. 2.6 PCR Amplification and Sequencing of the gyrB Gene 2.10 Analysis of the Nucleotide Sequences

Nearly complete DNA sequences of the gyrB gene The primary comparative analysis of the DNA

Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria 33 sequences determined in this study and represented in convenient and accurate tool to discriminate the the Gen Bank database was carried out using the majority of established species in the genus National Center for Biotechnology Information’s Xanthomonas [46], four close-related species were not Basic Local Alignment Search Tool (NCBI BLAST) supported with high branching level. [41]. Sequence alignment was performed using the As it was shown before, comparative analysis of the Clustal W 1.75 v software [42]. Based on nucleotide DNA sequences of the Xcc0006-0007 operon (length data, the phylogenetic trees were concatenated and 1,971 bp, 86.30% per whole length) is sufficient to constructed with the neighbor joining (NJ) [43] and minimum evolution (ME) [44] methods of MEGA 3.1 software [45].

3. Results and Discussion

3.1 Genus Xanthomonas

All bacterial cultures with different biochemical characteristics were tested on the corresponded test plants and produced either systemic or leaf spot lesions under glasshouse conditions. According to the 16S rRNA analysis, all strains were reliably (> 97% (a) similarity with type strain ATCC 33913T) identified as Xanthomonas spp.. After confirmation that investigated strains were affiliated to the same genus, DNA sequences of the gyrB gene [35] were sequenced (2,395 bp, 97.96% of whole its length) for all of them. Through cluster analysis (Fig. 1a), eight clades were branched with high bootstrap values (> 70%). Seven clades robustly comprised seven species (X. campestris, X. gardneri, X. translucence, X. oryzae, X. (b) arboricola, X. vesicatoria and X. albilineans). Seven Fig. 1 NJ tree of the nucleotide sequences for the gyrB strains were poorly discriminated. They formed the gene (a) and hin-region (b), based on 115 strains of one clade together with other X. euvesicatoria, X. Xanthomonas. The scale corresponds to the number of substitutions per 100 perforance, X. citri, X. fuscans and X. axonopodis base pairs (genetic distances). The numerals show statistical reference strains, and probably were just synonyms. reliability of the branching order (%) defined by bootstrap These data were supported by recent genomic analysis (1,000 replicas). Bootstrap values less 70% are not comparative analysis [26] and MLST analysis [21]. shown. The numbers in parentheses shows the amount of the investigated strains. For example, it had been hypothesized that X. citri and X. fuscans could belong to a single species [26]. Comparison of the gyrB DNA sequences revealed high variability for intra- (similarity values were greater than 79.3%) and inter-species (> 97.5%) levels Fig. 2 Schematic structures of the hin-regions of X. for Xanthomonas spp.. campestris, genotype hin I corresponding to the type strain Despite the fact that this analysis is considered as a ATCC 33913T.

34 Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria distinguish close-related Xanthomonas spp. strains below data exposed hin-region as a potential marker for the species level [36]. Based on the nucleotide diagnostics of comprehensive genus Xanthomonas at polymorphism and pathovar-specific deletions in the inter-species level. intergenic region (IR) (Fig. 2), robust species and 3.2 Genus Ralstonia inter-specific identification had been provided for X. campestris pv. campestris (referent strain, the length After inoculation of tomato and geranium test of the IR—140 bp), X. campestris pv. raphani (2 bp plants and analysis of complete DNA sequences of the deletion, type I), X. vesicatoria (25 bp deletion, type 16S rRNA gene (Gene Bank numbers are IIa), X. gardneri (27 bp deletion, type IIb), X. oryzae KJ606332-KJ606343), it had been shown that all pv. oryzae (35 bp, type IIIa), X. oryzae pv. oryzicola studied strains belonged to the R. solanacearum (Rs) (36 bp, type IIIb), and X. arboricola (7 bp, type IV). species complex (> 99.98% similarity with type strain The cluster analysis of the DNA sequences (data not Rs K60), race 3, biovar 2 (R3B2). Through the shown) had revealed six major groups, authentically. analysis of the gyrB gene, we authentically identified Five of them corresponded to the previously revealed all strains as Rs, phylotype IIB [22]. They were species. As it was shown by 16S rRNA, gyrB and grouped together with strains isolated from temperate whole-genome sequence analysis, X. axonopodis (9 bp region all over the world [47-49]. The gyrB nucleotide deletion, type V), X. citri (type V) and X. fuscans (V) sequences were 100% identical to the DNA sequences formed the single clade based on the 100% of the strain UW551, introduced long time ago to the homologous IR sequences. X. albilineans and X. cold regions of USA and Europe from Kenya [49]. transluceans did not contain Xcc0006 gene and IR. The saAFLP analysis of the strains revealed the same The results obtained by saAFLP correlated with the pattern with UW551 and was specific for phylotype previously defined topology. We robustly revealed IIB. The saAFLP pattern was comprised of 32 DNA eight species (as for gyrB), three subgroups for X. fragments. To design specific primers for PCR campestris, according to the pathovars pv. campestris amplification and sequencing of the phylotype II and pv. raphani; two subgroups for X. oryzae (pv. hin-regions, we used whole-genome sequences of oryzae, pv. oryzicola); two subgroups for X. Ralstonia sp. (Table 1) and related bacteria arboricola. Cupriavidus spp. retrieved from Gene Bank. The Then, we sequenced and provided cluster analysis primers (5'-CTTCGCAGCGTAGACGGAAAС-3' for the hin-region that, possibly, played the role into (RsF79) and 5'-CTTTGCAATGCGCTTCTGATG-3' host-plant specificity and virulence capacity [39] (Fig. (RsR715)) were tested for the specificity on the 1b). The schematic structure of the hin-region is wide-range sampling of Ralstonia spp. strains. The shown in Fig. 2. 566 bp PCR product (R1 located between the 1st and At the intra-species level, hin-region (R1) 2nd tRNA (Glu) genes) was revealed only for the sequences were unique, but at the inter-species level, strains of the phylotype II, R3B2. The determined the sequences identities were 30%-98%. On average, hin-region sequences were compared to the six substitutions were found for the strains belonging corresponding sequences of the other phylotypes to the same pathovar. Based on the hin-region retrieved from Gene Bank. The amount of specific structure, all strains were reliably delineated into eight substitutions for phylotype I was three, for phylotype distinct hin genotypes (I-VII).These groups correlated IIA-6, IIB-13, IV-4. The average sequence variability with the species and pathovar origins, in other words, (15.8%, including indels) exceeded the 16s rRNA reflected the practical taxonomic classification. The (3.7%) and gyrB (8.2%) ones. These values could be

Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria 35 understated partially due to inclusion of relatively small compared to mutS gene [22, 50], indicating the sample size. Using saAFLP and hin-region, better potential of these two approaches for diagnostics of separation of closely related Rs strains was obtained four phylotypes (Table 2).

Table 1 Presence of tRNA (Glu) copies and structure of hin-region in the whole sequenced genomes of the strains of genera Ralstonia and Sinorhizobium. Strain CTC* TTC* tRNA structure Begin** End** Role 1451200 1451275 1451466 1451540 S. fredii NGS234 1 3 (Glu) 4 s 1455542 1455616 1455835 1455909 1531855 1531930 1532147 1532222 S. medicae WSM419 1 3 (Glu) 4 s 1532279 1532353 1532539 1532614 1862108 1862183 1862401 1862476 S. meliloti 1021 1 3 (Glu) 4 s 1862534 1862608 1862790 1862865 2339565 2339640 R. eutropha JMP134 0 2 (Ala-Glu-Asp) 2 n/p 2340159 2340234 5012672 5012747 R. solanacearum UW551 0 2 (Ala-Glu-Asp) 2 p/p 5013162 5013237 1241767 1241842 R. solanacearum GMI1000 0 2 (Ala-Glu-Asp) 2 p/p 1242236 1242311 2236101 2236026 R. solanacearum CFBR2957 0 2 (Ala-Glu-Asp) 2 p/p 2236591 2236516 2308786 2308711 R. solanacearum PCI07 0 2 (Ala-Glu-Asp) 2 p/p 2309250 2309175 343062 343137 R. syzygii R24 0 2 (Glu) 2 p/p 343526 343601 *Anticodon; **tRNA (Glu) positions; s—symbiont; p/p—plant pathogen; n/p—non pathogen.

Table 2 The resolutions of the methods to study the biodiversity of the genera Sinorhizobium, Ralstonia and Xanthomonas. Methods Genus Species Phylotype/Pathovar Strain/Sequevar Phenotypic methods Biochemical analysis + +/- - +/- Inoculation/nodulation + +/- +/- - Genotypic methods 16S rRNA + +/- - - 16S-23S rRNA + + - - gyrB + + - - Xcc0006-0007* + + + - mutS** + +/- N/A - nodD*** + +/- +/- - egl** + + N/A +/- saAFLP - + + +/- hin-region PCR + + + - *only for genera Xanthomonas; **only for R. solanacearum; ***only for genus Sinorhizobium; N/A—not applicable.

36 Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria

3.3 Genus Sinorhizobium/Ensifer Group homology with whole-genome sequences of other species or genera. The topology of NJ tree was similar Nine nitrogen-fixing fast-growing strains to the data obtained before [54, 55]. The strains U1-3 Sinorhizobium spp., prospective for and S1-3 were robustly clustered together with the commercialization as a microbial seed inoculant to strains HH103 (97%-99% identity) and ATCC 35423T, soybeans seeds prior to planting, were studied in this which are able to nodulate soybeans. These data were work. These strains were isolated from nodules of the supported by inoculation test, which showed the different cultivars of soybeans (“Ustya”, “Svetlaya”, occurrence of wide-host range for these strains. Beside Glycine soja) in Russia in 2013. On the basis of the G. soja and G. max, they were able to effectively 16S rRNA sequences, biochemical characteristics and nodulate Phaseolis vulgaris and Vigna sp. The strains host specificity, nine investigated strains were Gs1-3 formed separate clusters closely related to attributed to the genus Ensifer. Based on the 16S USDA257. The sequence variability of the hin-regions rRNA sequence analysis, all strains were clustered for species S. fredii was ranging from 0% to 10.5%, together with S. americanum, S. fredii and S. which was reflected in the high separability values. xinjiangense, the last is considered as heterotypic synonym of S. fredii [51]. Thus, 16S rRNA gene 4. Conclusions sequence analysis lacked resolving power at species Summarizing all data below, we concluded that (or genomovars) level and below (genus variability < only integrated approach should be applied for 0.5%) [52]. The gyrB nucleotide sequences of the identification and diagnostics of genera with intricate studied strains were 97%-100% identical to the type taxonomic structure. This can be based on the strain S. fredii ATCC 35423T. The “Ustya” strains combination of phenotypic and genotypic tools. (U1, U2 and U3) were 100% identical to the ATCC Genotypic approach should include the analysis of 35423T. The strains isolated from G. soja (Gs1, Gs2 more than one phylogenetic marker gene, or methods and Gs3) formed separate branch and were closely with different resolution. Thus, we suggested: (1) 16S related to the broad-host-range strains USDA257 rRNS or gyrB for analysis of bacteria at the (enter symbiosis with wild soybean cultivars and some genera-species level; (2) hin-region and saAFLP for agronomical soybean cultivars) and NGR234 (unable determination of the inter-species variability. to enter symbiosis with any soybean). The strains Furthermore, hin-region PCR may be applied singly from “Svetlaya” (S1, S2, S3) formed a tight cluster as a rapid, efficient and low-cost method for direct with HH103 (forms nodules on agronomically monitoring and diagnostics of the species in the advanced soybean cultivars) [53]. The sequences were investigated genera. highly similar between all Ensifer species, and their variability for the genus saAFLP analysis, ranging Acknowledgments from 0% to 2.1%, revealed three types of saAFLP This work was supported by the programme patterns correlated with host plant of studied strains. “support of young scientists” by Presidium Russian Based on the conservative regions of the tRNA (Glu) Academy of Science. The authors are grateful to genes, the specific primers for the genus were Vasily Zotov and Sofia Khapchaeva for their technical developed. Nearly 300 bp PCR fragments (R1) were assistance. sequenced and compared to the corresponding sequences from Gene Bank. The R1 sequences were References unique for the S. fredii group and did not reveal any [1] De La Fuente, L., and Burdman, S. 2011. “Pathogenic

Identification and Diagnostics of Plant-Symbiotic and Phytopathogenic Bacteria 37

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